The modulation of heterochromatin structure and function plays a critical role in regulating gene expression, and defects in heterochromatin establishment and maintenance are associated with cancer, neural degeneracies, developmental pathologies, and other human diseases. However, many basic aspects of heterochromatin structure are poorly understood and are variously associated with conflicting models. This is due in large measure to experimental limitations. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures and dynamics of specific regulated chromatin domains in single cells. We have developed model systems using super-resolution localization microscopy through which we can study specific epigenetic chromatin structures at nanoscale resolution. To date we have successfully applied this system to visualizing active chromatin domains by probing patterns of combinatorial lysine acetylations. Here we propose to extend this approach to develop genetically encoded bioprobes suitable for super-resolution microscopy that will recognize epigenetic features of heterochromatin and DNA damage repair foci. Our preliminary results support a focus on two tudor domain motifs and the research proposed here thus focuses on two principal aims: (1) We will exploit the properties of fluorescent probes based on the tandem tudor domain of Setdb1. Based on current epifluorescence images, we hypothesize that the structures seen reflect the properties of heterochromatin. We will visualize the morphologies of bound chromatin structures, measure their dimensions and densities, and compare the structures observed at the nuclear periphery, the perinucleolar compartment, and internal nucleoplasm. We will test the prediction that our reporters colocalize with known epigenetic marks of heterochromatin and with heterochromatin protein components. We will also test the prediction that reporter-bound chromatin responds in parallel with experimental perturbation of heterochromatin structures. (2) We will develop novel genetically encoded fluorescent probes based on the tandem tudor domain of UHRF1. We will visualize the morphologies of bound chromatin structures, measure their dimensions and densities, and compare the properties of structures within the heterochromatin subcompartments. We will manipulate these probes for multivalent recognition of H3K9me3 and the free N-terminal end of H3 by combining the tandem tudor domain and PHD domains for combinatorial recognition. Together, these aims will result in the development and characterization of novel probes for the super-resolution visualization of critical epigenetic heterochromatin environments labeled in situ. These fundamental advances, in turn, have the potential to radically improve strategies for drug discovery, and yield new treatment modalities for pathologies associated defects in chromatin and epigenetic regulation.